Global surveillance of potential antiviral drug resistance in SARS-CoV-2: proof of concept focussing on the RNA-dependent RNA polymerase

Antiviral treatments for COVID-19 have involved many repurposed drugs. Currently, SARS-CoV-2 RNA-dependent RNA polymerase (RdRp, encoded by nsp12-nsp7-nsp8) has been targeted by numerous inhibitors with debated clinical impact. Among these, remdesivir has been conditionally approved for the treatment of COVID-19 patients. Although the emergence of antiviral resistance, an indirect proxy for antiviral efficacy, poses a considerable healthcare threat, an evolutionary perspective on emerging resistant mutants is still lacking. Here we show that SARS-CoV-2 RdRp is under purifying selection, that potential escape mutations are rare, and unlikely to lead to viral fitness loss. In more than 56,000 viral genomes from 105 countries dating from December 2019 to July 2020 we found negative selective pressure affecting nsp12 (Tajimas D = -2.62), with potential antiviral escape mutations in only 0.3% of sequenced genomes. Those affected known key residues, such as Nsp12:Val473 and Nsp12:Arg555. Of the potential escape mutations found globally, in silico structural models show that this rarely implies loss of stability in RdRp. No potential escape mutation were found in our local cohort of remdesivir treated patients from the first wave (n=8). Our results indicate that RdRp is a suitable drug target, and that remdesivir does not seem to exert high selective pressure. Our study could be the starting point of a larger monitoring effort of drug resistance throughout the COVID-19 pandemic. We recommend the application of repetitive genome sequencing of SARS-CoV-2 from patients treated with antivirals to provide early insights into the evolution or antiviral resistance.

Epidemiology and precision of SARS-CoV-2 detection following lockdown and relaxation measures

Introduction: SARS-CoV-2-detection is critical for clinical and epidemiological assessment of the ongoing CoVID-19 pandemic. Aim: To cross-validate manual and automated high-throughput (Roche-cobas6800-Target1/Target2) testing for SARS-CoV-2-RNA, to describe detection rates following lockdown and relaxation, and to evaluate SARS-CoV-2-loads in different specimens. Method: The validation cohort prospectively compared Basel-S-gene, Roche-E-gene, and Roche-cobas6800-Target1/Target2 in 1344 naso-oropharyngeal swabs (NOPS) taken in calendar week 13 using Basel-ORF8-gene-assay for confirmation. Follow-up-cohort-1 and -2 comprised 12363 and 10207 NOPS taken over 10 weeks until calendar week 24 and 34, respectively. SARS-CoV-2-loads were compared in follow-up NOPS, lower respiratory fluids, and plasma. Results: Concordant results were obtained in 1308 cases (97%) including 97 (9%) SARS-CoV-2-positives showing high quantitative correlations (Spearman r>0.95; p<0.001) for all assays. Discordant samples (N=36) had significantly lower SARS-CoV-2-loads (p<0.001). Following lockdown, weekly detection rates declined to <1% reducing single-test positive predictive values from 99.3% to 85.1%. Following relaxation, rates flared up to 4% with similarly high SARS-CoV-2-loads, but patients were significantly younger than during lockdown (34 vs 52 years, p<0.001). SARS-CoV-2-loads in follow-up NOPS declined by 3log10 copies/mL within 10 days post-diagnosis (p<0.001). SARS-CoV-2-loads in NOPS correlated weakly with those in time-matched lower respiratory fluids and plasma, but remained detectable in 14 and 7 cases of NOPS with undetectable SARS-CoV-2, respectively. Conclusion: Evaluated manual and automated assays are highly concordant and correlate quantitatively. Following successful lockdown, declining positive predictive values require dual-target-assays for clinical and epidemiologic assessment. Confirmatory and quantitative follow-up testing should be considered within <5 days, using lower respiratory fluids in symptomatic patients with SARS-CoV-2-negative NOPS.

SARS-CoV-2 phylogeny during the early outbreak in the Basel area, Switzerland: import and spread dominated by a single B.1 lineage variant (C15324T)

BackgroundThe first case of SARS-CoV-2 in Basel, Switzerland, was detected on February 26th 2020. We present a phylogenetic longitudinal study and explore viral introduction and evolution during the exponential early phase of the local COVID-19 outbreak from February 26th until March 23rd. MethodsWe sequenced SARS-CoV-2 from naso-oropharyngeal swabs, generated 468 high quality genomes, and called variants with our COVID-19 Pipeline (COVGAP). We analysed viral genetic diversity using PANGOLIN taxonomic lineages. To identify introduction and dissemination events we incorporated global SARS-CoV-2 genomes and inferred a time-calibrated phylogeny. FindingsThe early outbreak in Basel was dominated by lineage B.1 (83{middle dot}6%), detected from March 2nd, although the first lineage identified was B.1.1. Within B.1, a clade containing 68{middle dot}2% of our samples, defined by the SNP C15324T, suggests local spreading events. We infer the geographic origin of this mutation to our tri-national region. The remaining genomes map broadly over the global phylogenetic tree, evidencing several events of introduction from and/or dissemination to other regions of the world. We also observe family transmission events. InterpretationA single lineage dominated the outbreak in the City of Basel while other lineages such as the first (B1.1) did not propagate. Thus spreading events seem to have contributed most to viral spread, while travel returners and family transmissions were better controlled by the recommended measures. This phylogenetic analysis enriches epidemiological and contact tracing data, allowing connection of seemingly unconnected events, and can inform public health interventions. FundingNo dedicated funding was used for this work.

Effect of Systemic Inflammatory Response to SARS-CoV-2 on Lopinavir and Hydroxychloroquine Plasma Concentrations

BackgroundCoronavirus disease 2019 (COVID-19) leads to inflammatory cytokine release, which can downregulate the expression of metabolizing enzymes. This cascade affects drug concentrations in the plasma. We investigated the association between lopinavir (LPV) and hydroxychloroquine (HCQ) plasma concentrations and the values of acute phase inflammation marker C-reactive protein (CRP). MethodsLPV plasma concentrations were prospectively collected in 92 patients hospitalized at our institution. Lopinavir/ritonavir was administered 12-hourly, 800/200 mg on day 1, and 400/100 mg on day 2 until day 5 or 7. HCQ was given at 800 mg, followed by 400 mg after 6, 24 and 48 hours. Hematological, liver, kidney, and inflammation laboratory values were analyzed on the day of drug level determination. ResultsThe median age of study participants was 59 (range 24-85) years, and 71% were male. The median duration from symptom onset to hospitalization and treatment initiation was 7 days (IQR 4-10) and 8 days (IQR 5-10), respectively. The median LPV trough concentration on day 3 of treatment was 26.5 g/mL (IQR 18.9-31.5). LPV plasma concentrations positively correlated with CRP values (r=0.37, p<0.001), and were significantly lower when tocilizumab was preadministrated. No correlation was found between HCQ concentrations and CRP values. ConclusionsHigh LPV plasma concentrations were observed in COVID-19 patients. The ratio of calculated unbound drug fraction to published SARS-CoV-2 EC50 values indicated insufficient LPV concentrations in the lung. CRP values significantly correlated with LPV but not HCQ plasma concentrations, implying inhibition of cytochrome P450 3A4 (CYP3A4) metabolism by inflammation.